Development of a V. cholerae LPS (lipopolysaccharide)- based cholera vaccine is justified by the correlation of vibriocidal anti-LPS responses with immunity. Two O1 (serogroup) LPS serotypes, Inaba and Ogawa are associated with endemic and pandemic cholera. Both LPS serotypes induce protective antibody following infection or vaccination. Structurally, the serotypes are identical except for the terminal sugar (perosamine) which has a methoxyl group at position 2 in Ogawa, but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B cell epitope. Synthetic perosamine hexasaccharides of V. cholerae, serotype Ogawa coupled to BSA (CHO-BSA) induced protective antibody. We hypothesized that the terminal sugar of Inaba LPS would also induce protective antibody. Inaba neoglycoconjugates did induce antibodies but surprisingly they were not protective. In contrast, immunization with purified native Inaba LPS induced highly protective antibody. Preliminary data indicate that Inaba antibodies induced by the Inaba CHO-BSA conjugates have sufficient affinity to score in an ELISA but not enough to be protective in vivo. Priming with native LPS and then providing the Inaba CHO-BSA resulted in high ELISA titers in 10 days and antibodies that are protective. We hypothesize that Inaba LPS and Inaba CHO-BSA select different B cells, and that Inaba CHO-BSA can cross react with Inaba LPS-selected B cells to expand them and thus may provide an effective vaccine immunogen for those living in endemic areas with memory B cells but waning humoral immunity. Monoclonal antibodies (mAb) will be generated to the two Inaba immunogens to determine if different B cells are selected during immunization. An R21 grant is well suited for these studies that require reagent development to address a fundamental question in cholera biology. A new technology, surface plasmon resonance (SPR) will be used to characterize the affinity of the mAbs. The experiments are risky as they require generation of multiple reagents to complete the population analysis of anti-LPS antibodies. The study is high pay off because reagents, regardless of the exact specificity, will be generated that will map the antibody repertoire for Inaba LPS's terminal sugar. We will clarify the "type" of anti-V. cholerae LPS antibodies that are protective and thus substantiate or not the use of Inaba neoconjugates as a vaccine component.